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Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy.

Zhang T, Wang RY, Bao QY, Rawson DM - Theriogenology (2006)

Bottom Line: The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility.Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants.The results agreed well with those obtained from conventional volumetric based studies.

View Article: PubMed Central - PubMed

Affiliation: Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK. tiantian.zhang@luton.ac.uk

ABSTRACT
Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.

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Effect of cryoprotectant on impedance change. Six embryos were exposed to METH and DMSO at frequency 103.14 Hz over 30 min. (a) 1.0 M and (b) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.
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fig5: Effect of cryoprotectant on impedance change. Six embryos were exposed to METH and DMSO at frequency 103.14 Hz over 30 min. (a) 1.0 M and (b) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.

Mentions: Fig. 5 shows impedance values of six embryos exposed to METH and DMSO (1.0 and 2.0 M) at optimum frequency of 103.14 Hz over 30 min. The results showed that significant differences of impedance values were found between embryos in egg water and embryos in cryoprotectants. Comparisons of impedance changes during embryo exposure to 1.0 M METH and DMSO showed that the impedance values changed faster when embryos were exposed to METH than DMSO (Fig. 5a), indicating zebrafish embryos membrane permeability to 1.0 M METH is higher than to 1.0 M DMSO. Similar results were also found for 2.0 M concentrations (Fig. 5b). Statistical analysis also showed that the higher concentrations of cryoprotectants resulted in more significant change of impedance values. When embryos were exposed to 0.5 M METH or DMSO, no significant impedance changes were found over 30 min; for 1.0 M METH or DMSO exposure, significant impedance changes were found after 15 and 20 min, respectively; for 2.0 and 3.0 M METH exposure, significant impedance changes were detected after 3 min and for 2.0 M DMSO exposure, significant impedance changes were detected after 10 min.


Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy.

Zhang T, Wang RY, Bao QY, Rawson DM - Theriogenology (2006)

Effect of cryoprotectant on impedance change. Six embryos were exposed to METH and DMSO at frequency 103.14 Hz over 30 min. (a) 1.0 M and (b) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851733&req=5

fig5: Effect of cryoprotectant on impedance change. Six embryos were exposed to METH and DMSO at frequency 103.14 Hz over 30 min. (a) 1.0 M and (b) 2.0 M. The impedance values were normalised with respect to those obtained in egg water.
Mentions: Fig. 5 shows impedance values of six embryos exposed to METH and DMSO (1.0 and 2.0 M) at optimum frequency of 103.14 Hz over 30 min. The results showed that significant differences of impedance values were found between embryos in egg water and embryos in cryoprotectants. Comparisons of impedance changes during embryo exposure to 1.0 M METH and DMSO showed that the impedance values changed faster when embryos were exposed to METH than DMSO (Fig. 5a), indicating zebrafish embryos membrane permeability to 1.0 M METH is higher than to 1.0 M DMSO. Similar results were also found for 2.0 M concentrations (Fig. 5b). Statistical analysis also showed that the higher concentrations of cryoprotectants resulted in more significant change of impedance values. When embryos were exposed to 0.5 M METH or DMSO, no significant impedance changes were found over 30 min; for 1.0 M METH or DMSO exposure, significant impedance changes were found after 15 and 20 min, respectively; for 2.0 and 3.0 M METH exposure, significant impedance changes were detected after 3 min and for 2.0 M DMSO exposure, significant impedance changes were detected after 10 min.

Bottom Line: The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility.Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants.The results agreed well with those obtained from conventional volumetric based studies.

View Article: PubMed Central - PubMed

Affiliation: Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK. tiantian.zhang@luton.ac.uk

ABSTRACT
Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.

Show MeSH
Related in: MedlinePlus